Isolation, Culture, and Immunostaining of Skeletal Muscle Myofibers from Wildtype and Nestin-GFP Mice as a Means to Analyze Satellite Cells

  • Pascal Stuelsatz
  • Paul Keire
  • Zipora Yablonka-ReuveniEmail author
Part of the Methods in Molecular Biology book series (MIMB, volume 1556)


Multinucleated myofibers, the functional contractile units of adult skeletal muscle, harbor mononuclear Pax7+ myogenic progenitors on their surface between the myofiber basal lamina and plasmalemma. These progenitors, known as satellite cells, are the primary myogenic stem cells in adult muscle. This chapter describes our laboratory protocols for isolating, culturing, and immunostaining intact myofibers from mouse skeletal muscle as a means for studying satellite cell dynamics. The first protocol discusses myofiber isolation from the flexor digitorum brevis (FDB) muscle. These short myofibers are plated in dishes coated with PureCol collagen (formerly known as Vitrogen) and maintained in a mitogen-poor medium (± supplemental growth factors). Employing such conditions, satellite cells remain at the surface of the parent myofiber while synchronously undergoing a limited number of proliferative cycles and rapidly differentiate. The second protocol discusses the isolation of longer myofibers from the extensor digitorum longus (EDL) muscle. These EDL myofibers are routinely plated individually as adherent myofibers in wells coated with Matrigel and maintained in a mitogen-rich medium, conditions in which satellite cells migrate away from the parent myofiber, proliferate extensively, and generate numerous differentiating progeny. Alternatively, these EDL myofibers can be plated as non-adherent myofibers in uncoated wells and maintained in a mitogen-poor medium (± supplemental growth factors), conditions that retain satellite cell progeny at the myofiber niche similar to the FDB myofiber cultures. However, the adherent myofiber format is our preferred choice for monitoring satellite cells in freshly isolated (Time 0) myofibers. We conclude this chapter by promoting the Nestin-GFP transgenic mouse as an efficient tool for direct analysis of satellite cells in isolated myofibers. While satellite cells have been often detected by their expression of the Pax7 protein or the Myf5nLacZ knockin reporter (approaches that are also detailed herein), the Nestin-GFP reporter distinctively permits quantification of satellite cells in live myofibers, which enables linking initial Time 0 numbers and subsequent performance upon culturing. We additionally point out to the implementation of the Nestin-GFP transgene for monitoring other selective cell lineages as illustrated by GFP expression in capillaries, endothelial tubes and neuronal cells. Myofibers from other types of muscles, such as diaphragm, masseter, and extraocular, can also be isolated and analyzed using protocols described herein. Collectively, this chapter provides essential tools for studying satellite cells in their native position and their interplay with the parent myofiber.

Key words

Skeletal muscle Satellite cells Isolated myofiber Flexor digitorum brevis Extensor digitorum longus Diaphragm Masseter Extraocular muscles Pax7 MyoD Myogenin Nestin-GFP Myf5nLacZ MLC3F-nLacZ 3F-nlacZ-2E 



The methods and results summarized in the current chapter reflect the culmination of continuous updates during many years of research. Our current research is supported by grants to Z.Y.R. from the National Institutes of Health (AG035377, NS090051, NS088804). Pascal Stuelsatz is supported by an AFM-telethon fellowship (#18574). The authors are additionally grateful to the granting agencies (MDA, AHA, USDA-NRI, NIH) that have funded this research in the past and to our former members of our laboratory (Antony Rivera, Gabi Shefer, Andrew Shearer, and Elena Danoviz) for their valuable contributions.


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Authors and Affiliations

  • Pascal Stuelsatz
    • 1
  • Paul Keire
    • 1
  • Zipora Yablonka-Reuveni
    • 1
    Email author
  1. 1.Department of Biological Structure, School of MedicineUniversity of WashingtonSeattleUSA

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